This invention relates to the production of branched dendrimeric structures using phosphoramidite chemistry, and to the uses of the dendrimers.
Since their first synthesis, dendrimers have attracted considerable attention as a new branch of polymer science. Two basic strategies for the synthesis of these structures have been proposed: divergent, with the structure grown up from the centre to the periphery; and convergent, growth of the molecule from the periphery to the centre.
One of the most important parameters governing a dendrimeric structure and its generation, is the number of branches generated at each step; this defines the number of repetitive steps necessary to build up the desired molecule and the density of the groups at the periphery. The main properties of the dendrimeric molecule are determined by the functional end groups of moieties on its outer shell. Many applications proposed for dendrimers exploit the high density and the large number of these groups.
For example, dendrimers with a positively charged outer surface interact strongly with nucleic acid, a property which has been used recently for the transport of nucleic acids through the membranes of living cells; see Boussif et al, Proc. Natl. Acad. Sci., USA, (1995):92:7297-7301.
Branched (dendrimeric) oligonucleotides can be used to amplify radioactive or fluorescent signals in hybridisation tests. Such amplification may be particularly important in in situ hybridisation and in the emerging techniques which exploit oligonucleotide arrays, where the signal is limited by the surface density of the oligonucleotides or the target molecule.
Phosphitamide reagents have been described which double the amount of reactive 5xe2x80x2-hydroxyl groups after each condensation step, thus giving 2n reactive OH groups after n condensations, and they have been used in combination with biotin, fluorescein, pyrene and other phosphitamide synthons for multiple 5xe2x80x2-labelling of oligonucleotides. However, all these compounds have a particular disadvantage in that they result in highly dense structure which results in a high concentration of reporter groups, leading to self-quenching of fluorescence.
The present invention provides new reagents and methods which allow the assembly of large, branched structures from simple but versatile building blocks. The invention discloses dendrimers obtainable by the reaction of a core molecule having at least two reactive groups sequentially with doubling, trebling or more, branching synthons and optionally also with non-branching synthons, to provide a multimeric structure having branched units and optionally also non-branched units, and a plurality of blocked or unblocked functional groups at the outer ends of the structure.
In particular, the present invention discloses a dendrimer, wherein the core molecule and/or the synthons have the formula
(P1)F1xe2x80x94L1xe2x80x94J[xe2x80x94L2xe2x80x94F2(P2)]n
wherein F1(P1) is an optionally-protected functional group; the n F2(P2) groups are the same or different optionally-protected functional groups reactive with a functional group F1; L1 and L2 are the same or different linkers; J is a junction group; and n is 1(a non-branching synthon), 2 (a doubling, branching synthon), 3 (a trebling, branching synthon) or more.